This year we have continued to identify novel mAbs in several formats as Fabs, scFvs and eAds against cancer-related proteins mostly for pediatric patients. These mAbs in different formats were tested for their activity against cancer cells in vitro and in vivo and used for development of novel approaches for multispecific targeting. Two of the mAbs in CAR format, against CD22 and mesothelin, continued to be tested in clinical trials. We have also characterized drugability of some mAbs including their propensity for aggregation. The major accomplishments are summarized below. 1) Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. We demonstrated that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies. 2) CD16A (FcgammaRIIIA) is an activating receptor mostly expressed on natural killer (NK) cells and monocytes/macrophages. It can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) through low-affinity interaction with human immunoglobulin G (IgG) Fc. It can also mediate cell lysis if NK cells are guided by bispecific killer cells engagers (BiKEs). BiKEs showed some success in clinical trials of cancer and are promising candidate therapeutics. However, currently reported BiKEs are based on antibody fragments (scFvs) of relatively large size. The CD16A-specific antibodies are also typically from animal origin. Decreasing the BiKE size could result in enhanced penetration into solid tumor and normal tissues, and using fully human antibodies could decrease the likelihood of immunogenicity. We identified and characterized two antibody domains, D6 and E11, isolated from a very large human VH antibody domain library displayed on phage. D6 and E11 bound CD16A with EC50 of 4nM and 8nM, respectively, but not other Fc gamma receptors (FcgammaRs) such as CD64 (FcgammaRI), CD32 (FcgammaRII) and CD16B (FcgammaRIIIB). They bound to both CD16A allotypes (158F,V) with equal affinity and competed with each other as well as with human IgG1 and the mouse anti-CD16A antibody 3G8. These and other results were used to build a molecular docking model predicting that D6 and E11 may bind to the CD16A membrane proximal D2 domain by interacting with its BC, C'E and EF loops. Importantly, cross-linked (bivalent) D6 and E11 induced secretion of IL-2 after binding to CD16A-expressing Jurkat T cells. The small size of these antibody domains combined with their high-affinity, specific, allotype-independent, activating interactions with CD16A could allow generation of novel highly effective BiKEs and other candidate protein therapeutics.Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. 3) Smaller recombinant antibody fragments are now emerging as alternatives of conventional antibodies. Especially, immunoglobulin (Ig) constant CH2 domain and engineered CH2 with improved stability are promising as scaffolds for selection of specific binders to various antigens. We constructed a yeast display library based on an engineered human IgG1 CH2 scaffold with diversified loop regions. A group of CH2 binders were isolated from this yeast display library by panningagainst nucleolin, which is a tumor-associated antigen involved in cell proliferation, tumor cell growth and angiogenesis. Out of 20 mutants, we selected 3 clones exhibiting relatively high affinities to nucleolin on yeasts. However, recombinant CH2 mutants aggregated when they were expressed. To find the mechanism of the aggregation, we employed computational prediction approaches through structural homology models of CH2 binders. The analysis of potential aggregation prone regions (APRs) and solvent accessible surface areas (ASAs) indicated two hydrophobic residues, Val264 and Leu309, in the beta-sheet, in which replacement of both charged residues led to significant decrease of the protein aggregation. The newly identified CH2 binders could be improved to use as candidate therapeutics or research reagents in the future. 4) Currently, we participate in the work of the Pediatric Oncology Dream team where we identified and performed initial characterization of several mAbs against targets of importance for childhood cancers. Some of the results were described above and others especially the mAbs against GPC2 will be reported in the next year report after additional characterization of those antibodies including in animal models.